Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

Antonella San Romerio; Giovanni Fiorillo; Ileana Terruzzi; Pamela Senesi; Giulio Testolin; Alberto Battezzati

doi:10.1016/j.ab.2004.09.034

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

Antonella San Romerio, Giovanni Fiorillo, Ileana Terruzzi, Pamela Senesi, Giulio Testolin, Alberto Battezzati

Research output: Contribution to journal › Article › peer-review

Abstract

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2- ¹³C]cytosine and [2- ¹³C]5- methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.

Original language	English
Pages (from-to)	158-163
Number of pages	6
Journal	Analytical Biochemistry
Volume	336
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2004.09.034
Publication status	Published - Jan 15 2005

Keywords

5-Methylcytosine
Cytosine
DNA methylation
Gas chromatography-mass spectrometry
Stable isotopes

ASJC Scopus subject areas

Biochemistry
Biophysics
Molecular Biology

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10.1016/j.ab.2004.09.034

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title = "Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry",

abstract = "A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2- 13C]cytosine and [2- 13C]5- methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.",

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AU - Romerio, Antonella San

AU - Fiorillo, Giovanni

AU - Terruzzi, Ileana

AU - Senesi, Pamela

AU - Testolin, Giulio

AU - Battezzati, Alberto

PY - 2005/1/15

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N2 - A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2- 13C]cytosine and [2- 13C]5- methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.

AB - A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2- 13C]cytosine and [2- 13C]5- methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.

KW - 5-Methylcytosine

KW - Cytosine

KW - DNA methylation

KW - Gas chromatography-mass spectrometry

KW - Stable isotopes

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Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

Abstract

Keywords

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